U.S. patent application number 13/653829 was filed with the patent office on 2013-02-21 for collagenous material.
This patent application is currently assigned to TISSUE SCIENCE LABORATORIES PLC. The applicant listed for this patent is TISSUE SCIENCE LABORATORIES PLC. Invention is credited to Paul ARMITAGE, Christine Elizabeth DAWSON.
Application Number | 20130045923 13/653829 |
Document ID | / |
Family ID | 47713068 |
Filed Date | 2013-02-21 |
United States Patent
Application |
20130045923 |
Kind Code |
A1 |
ARMITAGE; Paul ; et
al. |
February 21, 2013 |
COLLAGENOUS MATERIAL
Abstract
The present invention relates to a collagenous material and to a
process for the manufacture thereof. The process is for the
manufacture of a collagenous material from a plurality of collagen
particles, wherein said collagen particles are derived from a
natural tissue material and are substantially free of non-fibrous
tissue proteins, cellular elements and lipids or lipid residues,
and wherein said collagen particles comprise fragments of collagen
fibres displaying original collagen fibre architecture and
molecular ultrastracture of said natural tissue material, said
process comprising steps of treating the collagen particles with an
aqueous acid solution to swell the collagen particles; and
collectively dehydrating the swollen collagen particles. The
collagenous material may be used, for example, in wound care.
Inventors: |
ARMITAGE; Paul; (West
Yorkshire, GB) ; DAWSON; Christine Elizabeth; (West
Yorkshire, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TISSUE SCIENCE LABORATORIES PLC; |
West Yorkshire |
|
GB |
|
|
Assignee: |
TISSUE SCIENCE LABORATORIES
PLC
West Yorkshire
GB
|
Family ID: |
47713068 |
Appl. No.: |
13/653829 |
Filed: |
October 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13392965 |
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PCT/GB2010/001410 |
Jul 26, 2010 |
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13653829 |
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Current U.S.
Class: |
514/17.2 ;
435/68.1; 530/356 |
Current CPC
Class: |
A61P 17/02 20180101;
A61K 38/39 20130101; C07K 14/78 20130101 |
Class at
Publication: |
514/17.2 ;
530/356; 435/68.1 |
International
Class: |
A61K 38/39 20060101
A61K038/39; C12P 21/06 20060101 C12P021/06; A61P 17/02 20060101
A61P017/02; C07K 14/78 20060101 C07K014/78 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 2009 |
GB |
0915049.1 |
Claims
1-26. (canceled)
27. A process for the manufacture of a collagenous material from a
plurality of collagen particles, wherein the collagen particles are
derived from a natural tissue material and are substantially free
of non-fibrous tissue proteins, cellular elements and lipids or
lipid residues, and wherein the collagen particles comprise
fragments of collagen fibres displaying an original collagen fibre
architecture and molecular ultrastracture of natural tissue
material, the process comprising the steps of: treating the
collagen particles with an aqueous acid solution to swell the
collagen particles; and collectively dehydrating the swollen
collagen particles.
28. A process according to claim 27, wherein the collagen particles
are suspended in the aqueous acid solution.
29. A process according to claim 28, further comprising mixing the
collagen particles in the aqueous acid solution.
30. A process according to claim 29, wherein the mixing step
includes homogenising the collagen particles in the aqueous acid
solution.
31. A process according to claim 27, wherein the step of
collectively dehydrating the swollen collagen particles includes
treating the swollen collagen particles with a dehydrating
agent.
32. A process according to claim 31, wherein the dehydrating agent
comprises a water miscible solvent.
33. A process according to claim 32, wherein the water-miscible
solvent is selected from the group consisting of acetone, ethanol,
ether, and mixtures thereof.
34. A process according to claim 27, wherein the step of
collectively dehydrating the swollen collagen particles includes
air drying.
35. A process according to claim 27, further comprising
collectively shaping the swollen collagen particles prior to
dehydration.
36. A process according to claim 35, wherein the swollen collagen
particles are collectively shaped into a sheet.
37. A process according to claim 35, further comprising freezing
the collectively shaped swollen collagen particles.
38. A process according to claim 27, further comprising
cross-linking the swollen collagen particles prior to and/or
concurrently with dehydration.
39. A process according to claim 27, further comprising
cross-linking the collagenous material after dehydration.
40. A process according to claim 27, further comprising treating
the natural tissue material with an organic solvent and with a
proteolytic enzyme.
41. A process according to claim 40, wherein the proteolytic enzyme
comprises trypsin.
42. A process according to claim 27, further comprising rehydrating
the collagenous material.
43. A process for the manufacture of a collagenous sheet material
from collagen particles, wherein the collagen particles are derived
from a natural tissue material and are substantially free of
non-fibrous tissue proteins, cellular elements and lipids or lipid
residues, and wherein the collagen particles comprise fragments of
collagen fibres displaying an original collagen fibre architecture
and molecular ultrastracture of natural tissue material, the
process comprising the steps of: suspending the collagen particles
in an aqueous acid solution to swell the collagen particles;
collectively forming the swollen collagen particles into a sheet;
and dehydrating the sheet of swollen collagen particles.
44. A collagenous material obtainable by the process according to
claim 27.
45. A collagenous material obtainable by the process according to
claim 43.
46. A method of treatment of a wound comprising the step of
applying to the wound a collagenous material according to claim
44.
47. A method of treatment of a wound comprising the step of
applying to the wound a collagenous material according to claim
45.
48. A method of reducing bleeding from a body site comprising the
step of applying to the body site a collagenous material according
to claim 44.
49. A method of reducing bleeding from a body site comprising the
step of applying to the body site a collagenous material according
to claim 45.
Description
[0001] The present invention relates to a collagenous material and
to a process for the manufacture thereof. The collagenous material
may be used, for example, in wound care.
[0002] Collagen is a triple-helix protein which forms the major
part of the dermal extracellular matrix (ECM), together with
glycosaminoglycans, proteoglycans, laminin, fibronectin, elastin
and cellular components. The ECM is the largest component of the
dermal skin layer and the synthesis of ECM is a key feature of
wound healing, especially where there has been a significant loss
of tissue that precludes closure by primary intention.
[0003] The principal function of collagen in the dermal ECM is to
act as a scaffold in connective tissue. Predominantly, collagen is
present in the form of type I collagen (80-85%) and type III
collagen (8-11%), both of which are fibrillar or rod-shaped
collagens. The tensile strength of skin is due largely to these
collagen molecules assembling into fibrils, with adjacent molecules
crosslinking to further increase tensile strength. However,
collagen laid down during wound healing has a reduced structural
integrity compared to unwounded tissue; scar tissue rarely exceeds
70% of unwounded tissue strength.
[0004] In addition to being the main component of scar tissue,
collagen has a key role in the control of the inflammatory response
to injury and subsequent repair, with functions that influence
cellular mitogenesis, differentiation and migration; protein
synthesis in the ECM; synthesis and release of inflammatory
cytokines and growth factors; and interactions between enzymes
which remodel the ECM, including matrix metalloproteinases (MMPs)
and their tissue inhibitors (TIMPs).
[0005] For medical applications, purified fibrous collagen has long
been known to be a useful therapeutic adjunct to aid wound healing.
Research into collagen-based dressing materials has shown that
collagen significantly increases the production of fibroblasts in
wounds, encouraging direct migration of cells into the wound
enhancing the formation of new, organised collagen fibres.
[0006] Purified fibrous collagen pads have also been used to
provide haemostatic dressings. When in contact with a bleeding
surface, a fibrous collagen haemostat attracts platelets which
adhere to collagen fibrils and release coagulation factors. This
coagulation and aggregation of the platelets into thrombi on the
collagenous mass, provides the formation of a physiologic platelet
plug which slows and eventually stops the bleeding. The collagen
fibrils also provide a structural matrix strengthening the platelet
plug.
[0007] Previous studies have demonstrated that collagenous
materials prepared using a process that retains the original fibre
architecture and molecular ultrastructure of the natural tissues
from which they are derived have a number of advantageous
properties. For example, U.S. Pat. No. 5,397,353 discloses a
process for the preparation of collagenous materials that are
substantially non-antigenic and substantially free of non-fibrous
tissue proteins, cellular elements, lipids and lipid residues. The
materials are typically sheet structures and are useful, for
example, as implants. The collagenous sheet materials are
susceptible to colonisation and vascularisation by host cells
following implantation and are resistant to calcification.
[0008] It has further been shown that the favourable properties of
these collagen sheet materials can be retained when the collagen
material is presented in the form of particles comprising fibre
fragments. EP 1112096 describes an injectable or mouldable
composition of collagen fibre fragments prepared from the
relatively large-scale sheet materials of U.S. Pat. No. 5,397,353
in such a way as to retain the natural collagen fibre architecture
and molecular ultrastructure. This size reduction is achieved by
careful grinding or milling of the collagenous sheet materials,
which may then be suspended to form a paste or injectable
composition, as appropriate.
[0009] The present invention provides a novel collagenous material
particularly suitable for use as a wound dressing.
[0010] According to a first aspect of the present invention there
is provided a process for the manufacture of a collagenous material
from a plurality of collagen particles, wherein said collagen
particles are derived from a natural tissue material and are
substantially free of non-fibrous tissue proteins, cellular
elements and lipids or lipid residues, and wherein said collagen
particles comprise fragments of collagen fibres displaying original
collagen fibre architecture and molecular ultrastracture of said
natural tissue material, said process comprising steps of: [0011]
treating the collagen particles with an aqueous acid solution to
swell the collagen particles; and [0012] collectively dehydrating
the swollen collagen particles.
[0013] By dehydrating the mass of swollen collagen particles, a
coherent collagenous material is formed. The collagenous material
has excellent properties for use as a wound dressing or as a
component of a wound dressing. This porous, biocompatible material
is non-antigenic and of natural origin, and provides a
skin/tissue-like feel which improves patient acceptance and
satisfaction. The collagenous material is soft and conformable,
which is clearly advantageous for application to wound sites.
Significantly, the collagenous material has good inherent strength
and may be wrapped around wound areas to form an effective barrier.
The collagenous material maintains a moist wound environment to
promote wound healing, and its application helps to reduce
contraction and scar tissue formation. Unlike a number of existing
materials used in wound dressings, the collagenous material does
not gel on wound contact.
[0014] Further, the presence of collagen at a wound site is known
to increase the rate of wound healing.
[0015] Thus, the collagenous material described herein is
particularly useful in wound care. The collagenous material may be
presented in a hydrated state. The process may therefore further
comprise at least one rehydration step, in which the collagenous
material is maintained in an aqueous solution. For instance, the
collagenous material may be maintained in saline, such as 0.9%
saline.
[0016] The presentation of the collagenous material in a hydrated
form helps to maintain a moist wound environment when the material
is applied as a wound dressing. Surprisingly, it has been found
that the collagenous material formed from collagen particles
remains intact following processing/manufacture and does not return
to a particulate state following rehydration.
[0017] The coherent collagenous material produced using the process
of the present invention may be manufactured in any size, and can
therefore be used to provide coverage of large wound sites, such as
burns. The structural integrity of the collagenous material
provides good protection to a wound site and the may be readily
secured in position to prevent migration.
[0018] Although the processing typically results in some disruption
to the structure of the collagen, the disruption is less than in
prior art processes, which involve harsh chemical and mechanical
treatments that significantly disrupt the collagen substructure to
leave only collagen fragments in the form of tropocollagen (see,
for example, U.S. Pat. No. 3,157,524 and U.S. Pat. No. 4,412,947).
In contrast, collagenous materials manufactured according to the
process of the present invention show good retention of collagen
fibril structure and alignment, at least in part.
[0019] The natural tissue material may comprise any
collagen-containing tissue material of human or animal origin. The
natural tissue material may be a tissue comprising predominantly
type I collagen. Preferred starting materials include dermis and
tendons. In some embodiments, it is preferred that porcine tissue
materials are processed to provide the collagenous material,
although it will be understood that other mammalian sources may
alternatively be employed, such as, for example, primates, cows,
sheep, goats or horses.
[0020] Depending upon the starting material, the particles of
collagen may contain a proportion of elastin. Thus, the particles,
and the coherent collagenous materials formed therefrom, consist
essentially of collagen optionally with small proportions of
elastin.
[0021] The particles of acellular collagen may be formed using any
suitable process. The original collagen fibre architecture and
molecular ultrastructure of the natural tissue material must be
retained in the particles. Preferred processes for preparing the
collagen particles are therefore analogous to those described in EP
1112096, the contents of which are incorporated herein by
reference. Preferably, collagenous material prepared in the form of
a sheet or other large-scale structure is milled to form the
particles. The collagenous material may be prepared by a process
analogous to those described in U.S. Pat. No. 5,397,353, the
contents of which are incorporated herein by reference. The
collagenous tissue is neither solubilised nor denatured in the
process, so its natural structure is maintained.
[0022] Thus, freshly cut natural tissue material may be treated to
remove therefrom substantially all lipids and lipid residues and
thereafter treated to remove non-fibrous tissue proteins and
cellular elements.
[0023] The lipid extraction may be achieved by solvent extraction
using an organic solvent, such as acetone. Other non-limiting
examples of suitable solvents include non-aqueous solvents such as
ethanol and ether.
[0024] Non-fibrous tissue proteins include glycoproteins,
proteoglycans, globular proteins and the like. Cellular elements
include antigenic proteins and enzymes and other cellular debris
arising from the processing conditions. These portions of the
natural tissue material may be removed by treatment with a
proteolytic enzyme, such as trypsin. It has previously been found
that above 20.degree. C. treatment with trypsin can in some
circumstances result in an alteration of the collagen fibre
structure leading to a lower physical strength. Moreover, low
temperatures discourage the growth of microorganisms in the
preparation. It is therefore preferred to carry out the treatment
with trypsin at a temperature below 20.degree. C. Moreover, trypsin
is more stable below 20.degree. C. and lower amounts of it may be
required. Any suitable trypsin concentration may be used, for
instance a concentration within the range of around 0.01 g/l to 25
g/l. It has been found that good results can be obtained using
about 2.5 g/l trypsin.
[0025] Further treatments may optionally be carried out, such as
treatment with one or more additional enzymes, for example a
carbohydrate-splitting enzyme.
[0026] Those substances said to be "substantially free" of
materials generally contain less than 5% of, and preferably less
than 1%, of said materials.
[0027] The resulting collagenous material is then reduced to
particles, care being taken to ensure that the size reduction is
not associated with a degradation of the original collagen fibre
architecture and molecular ultrastructure of the starting material.
The particles may be produced by grinding or milling using, for
example, a ball or hammer mill, which may be cooled to an
appropriate temperature. The sheet material may be cut into small
pieces prior to milling. Milling may be carried out in dry form
(less than 10% moisture content) or in frozen hydrated form (20-80%
moisture content).
[0028] The collagen particles may be of any suitable size.
Typically, the collagen particles have a mean diameter within the
range of from around 5 .mu.m to around 1000 .mu.m, more typically
from around 50 .mu.m to around 500 .mu.m. Good results have been
achieved using collagen particles with a mean diameter of
approximately 150 .mu.m.
[0029] Any suitable aqueous acid solution may be used to treat the
collagen particles. The choice of acid employed is not critical
since the purpose is to lower the pH so that individual collagen
particles readily absorb water. A weak organic or inorganic acid
will generally be used. Non-limiting examples include dilute
hydrochloric acid, acetic acid, and boric acid. Particularly good
results have been observed using glacial acetic acid.
[0030] The concentration of the acid is typically at least 0.1% in
order to provide a suitable pH for swelling. Acid can have a
detrimental effect on the structure of collagen and therefore care
should be taken to ensure that the aqueous acid solution is not too
concentrated. The acid concentration should generally not exceed
1%. Typically, the pH is between 1 and 4, and more typically is
between 2 and 4. Particularly good results have been achieved at
around pH 3 to 3.5.
[0031] Typically, treatment of the collagen particles with the
aqueous acid solution involves suspending the particles in the
aqueous acid solution. The collagen particles may be weighed to
determine the amount of aqueous acid solution required. Typically,
solid collagen content may vary from about 0.5% to 5% of the
aqueous acid solution volume. For example, good results have been
observed using a formulation of 1% collagen in aqueous acid
solution. The collagen particles are treated with the aqueous acid
solution for sufficient time to allow swelling of the particles.
This may take up to 24 hours or more.
[0032] In preferred embodiments, however, the collagen particles
are suspended in the aqueous acid solution and mixed to accelerate
swelling and to give an even distribution thereby ensuring
relatively uniform end products.
[0033] Mixing can be achieved by various means, including mixing by
hand or using mechanical mixing apparatus. For example, mixing can
be carried out by homogenising, agitating, shaking, or blending.
Preferably, the collagen particles are distributed in the aqueous
acid solution by homogenisation.
[0034] Homogenisation of the suspension of swollen collagen
particles results in a dispersion in the form of a slurry or
`paste` of swollen collagen particles. If fully dispersed, the
combination of swollen collagen particles and aqueous acid solution
may be considered a colloid.
[0035] Typically, solid collagen content may vary from about 0.5%
to 5% of the solution volume. As a consequence of the reduced pH,
the collagen particles swell in the aqueous acid solution and the
increase in particle size reduces the fluidity of the dispersion.
At concentrations of, for example, around 0.5 to 1% the dispersion
can be easily mixed and poured. However, higher concentrations of
collagen become difficult to mix due to the considerable increase
in viscosity. At concentrations of about 2% and above, the
dispersion may be relatively difficult to mix and generally cannot
be poured.
[0036] In preferred embodiments, dehydration is carried out by
treatment of the swollen collagen particles with a dehydrating
agent. Any suitable dehydrating agent may be used. For example, the
dehydrating agent may comprise any water miscible solvent that does
not react with or dissolve collagen. Non-limiting examples of
suitable dehydrating agents include non-aqueous solvents such as
acetone, ethanol, ether, or mixtures thereof. The volume of solvent
required to dehydrate the mass of swollen collagen particles may
vary according to the solvent(s) used and the volume of collagen
present. By way of example, acetone may be used at a ratio of about
5:1 solvent to collagen by volume. Multiple solvent rinses may be
required to ensure complete removal of water. For instance, good
results have been achieved using 3.times.45-minute rinses with
acetone.
[0037] The solvent-dehydrated collagenous material is porous, with
a sponge-like appearance.
[0038] In alternative embodiments, the swollen collagen particles
may be dehydrated by air drying. For example, the mass of swollen
collagen particles may be left at room temperature to allow air
drying. Typically, air drying is carried out for at least about 24
hours, although for complete dehydration a period of about 48 hours
or more may be required.
[0039] The collagenous material dehydrated by air-drying is porous,
with a translucent, foam-like appearance which differs from the
solvent-dehydrated collagenous material.
[0040] Other means of dehydrating may also be used, such as vacuum
drying.
[0041] The process may optionally include a step of collectively
shaping the swollen collagen particles prior to dehydration, the
mass of swollen collagen particles then being dehydrated in the
selected shape or conformation. It has been found that the shape or
conformation of the dehydrated collagenous material is essentially
determined by the shape or conformation of the collection of
swollen collagen particles during the dehydration step. In other
words, dehydration to some extent `fixes` the shape or conformation
of the final collagenous material. Usefully, therefore, the swollen
collagen particles may be collectively shaped so that the resulting
shape or conformation of the mass of swollen collagen particles is
retained, or at least partly retained, in the collagenous material
after dehydration.
[0042] Typically, the swollen collagen particles are presented in
the form of a suspension, slurry or paste, in conjunction with the
aqueous acid solution. The swollen collagen particles may, for
example, he moulded, formed or cast into the desired shape prior to
dehydration.
[0043] In a particularly preferred embodiment, the swollen collagen
particles are formed into a sheet, film or similar shape. This may
be achieved by casting the swollen collagen particles or by placing
them into a suitable mould.
[0044] Other non-limiting examples of suitable shapes include
beads, ropes or variations thereof.
[0045] It is desirable to maintain the shape or conformation of the
collection of swollen collagen particles during dehydration.
Although shape or conformation may be held in any suitable manner,
freezing has been found to be a particularly convenient way to
maintain the shape or conformation before and during the
dehydration step. Therefore, after shaping, the collection of
swollen collagen particles may optionally be frozen. Any means of
freezing may be used. For instance, freezing may be carried out in
an industrial, laboratory or domestic freezer, or by blast freezing
or cryogenic freezing.
[0046] Optionally, cross-linking may be carried out to impart
additional physical strength to the collagenous material, and an
increased resistance to digestive enzymes that may be present in a
wound healing environment.
[0047] The swollen collagen particles may be cross-linked before
the dehydration step and/or concurrently therewith. Additionally or
alternatively, the collagenous material may be cross-linked after
dehydration.
[0048] Whilst any cross-linking agent may be used, preferred
cross-linking agents include polyisocyanates, in particular
diisocyanates. The polyfunctional isocyanates react with amino or
hydroxyl groups of different protein chains so forming a material
which has a stable structure retaining the original architecture of
the collagen and which is resistant to enzymatic attack. It is
known that antigenicity is associated with the amino groups of the
protein chains of collagen, and reacting the amino groups with
isocyanate removes any antigenicity associated with these groups.
Preferred diisocyantes include aliphatic, aromatic and alicyclic
diisocyanates as exemplified by 1,6-hexamethylene diisocyanate,
toluene diisocyanate, 4,4'-diphenylmethane diisocyanate, and
4,4'-dicyclohexylmethane diisocyanate, respectively. A particularly
preferred diisocyanate is hexamethylene diisocyanate (HMDI).
Carbodiimide cross-linking agents may also be used, such as
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC).
[0049] Where the cross-linking agent is used to treat
non-dehydrated collagen material, a surfactant may be used to
ensure proper dispersion of the cross-linking agent.
[0050] The extent of cross-linking may be varied. Usefully, this
provides a mechanism for controlling the rate at which the
collagenous material is resorbed or degraded during use. The
resistance to degradation tends to increase as the extent of
cross-linking is increased.
[0051] By way of example, cross-linking may be carried out using
HMDI. As a guide, the HMDI may be used at a concentration of around
0.01 g to 0.5 g per 50 g of collagen. If the concentration is too
high, this may result in over-cross-linking and foreign body
reactions. Cross-linking may be carried out for a range of
different time periods. By way of example, the collagen may be
exposed to the cross-linking agent for between around 1 hour and
around 3 days. Typically, cross-linking is carried out for at least
12 hours, preferably at least 20 hours.
[0052] It will be appreciated that the cross-linking conditions may
routinely be varied in order to adjust the extent of
cross-linking.
[0053] According to a further aspect of the present invention there
is provided a process for the manufacture of a collagenous sheet
material from collagen particles, wherein said collagen particles
are derived from a natural tissue material and are substantially
free of non-fibrous tissue proteins, cellular elements and lipids
or lipid residues, and wherein said collagen particles comprise
fragments of collagen fibres displaying original collagen fibre
architecture and molecular ultrastracture of said natural tissue
material, said process comprising steps of: [0054] suspending the
collagen particles in an aqueous acid solution to swell the
collagen particles; [0055] collectively shaping the swollen
collagen particles into a sheet; and [0056] dehydrating the sheet
of swollen collagen particles.
[0057] Optionally, the process may incorporate the additional steps
as described herein.
[0058] According to a further aspect of the present invention there
is provided a collagenous material obtainable by a process as
herein described.
[0059] A range of different factors may be added to the collagenous
material, such as growth factors, clotting agents, or other
pharmaceutically active agents. The collagenous material may be
seeded with cells, such as stem cells.
[0060] The collagenous material is useful in wound care. The
collagenous material may also be used as a haemostat, to reduce or
prevent blood flow from a body site.
[0061] According to a further aspect of the present invention there
is provided a collagenous material as herein described for use in
therapy.
[0062] According to a further aspect of the present invention there
is provided the use in therapy of a collagenous material as herein
described.
[0063] According to a further aspect of the present invention there
is provided a collagenous material as herein described for use in
wound care.
[0064] According to a further aspect of the present invention there
is provided the use in wound care of a collagenous material as
herein described.
[0065] According to a further aspect of the present invention there
is provided a method of treatment of a wound comprising the step of
applying to the wound a collagenous material as herein
described.
[0066] According to a further aspect of the present invention there
is provided a collagenous material as herein described for use as a
haemostat.
[0067] According to a further aspect of the present invention there
is provided the use in haemostasis of a collagenous material as
herein described.
[0068] According to a further aspect of the present invention there
is provided a method of reducing bleeding from a body site
comprising the step of applying to the body site a collagenous
material as herein described.
[0069] Embodiments of the present invention will now be described
further in the following non-limiting examples and with reference
to the accompanying drawing, in which:
[0070] FIG. 1 is a scanning electron micrograph at .times.10000
magnification of a representative sample of the collagenous
material according to the present invention.
EXAMPLES
1. Manufacture of Solvent-Dehydrated Collagenous Sheet Material
[0071] Acellular collagen sheet material was prepared according to
the method disclosed in U.S. Pat. No. 5,397,353 and reduced to
particles as described in EP 1112096.
[0072] Thus, freshly cut dermis harvested from sows was immersed in
acetone. After 1 hour, the acetone was removed and replaced by
fresh acetone. After a further incubation for around 36 hours, the
tissue was removed from the acetone and placed in 0.9% saline to
extract residual acetone. The tissue was then digested for 28 days
with a solution of trypsin at a concentration of 2.5 mg/ml in 0.1 M
phosphate buffer with 0.5 mg/ml sodium azide as a bacteriostatic
agent. The purified tissue was removed from the trypsin solution
and rinsed in buffer.
[0073] The resulting collagenous sheet material was cut into small
pieces (approximately 5 mm.times.10 mm in size) before being milled
cryogenically to a mean particle size of around 150 .mu.m.
[0074] The collagen particles were suspended in a 0.6% glacial
acetic acid solution such that a concentration of about 1% solid
collagen was formulated. This suspension was homogenised for about
30 seconds using a Silverson.RTM. L4RT homogeniser to evenly
disperse the swollen collagen particles throughout the suspension,
thereby forming a slurry or `paste` of swollen collagen
particles.
[0075] This dispersion was then cast into a pre-formed polystyrene
mould and frozen at -20.degree. C. in a laboratory freezer. Once
frozen, the material was removed from the mould and defrosted in
acetone to give a dehydrated collagenous sheet. Several acetone
rinses were performed to ensure complete dehydration of the
collagen.
[0076] The collagenous sheet material was then cross-linked using
HMDI in acetone. A concentration of about 0.1 g HMDI per 50 g of
solid collagen was added. The material was cross-linked for at
least 20 hours, rinsed in acetone, and allowed to air dry.
[0077] The collagenous material is porous, with a sponge-like
appearance.
[0078] Upon rehydration, the collagenous sheet material did not gel
or fragment, and had good structural integrity.
2. Manufacture of Air-Dried Collagenous Sheet Material
[0079] The process of Example 1 was followed up to the step of
casting the collagen dispersion into the pre-formed polystyrene
mould.
[0080] There followed a step of air-drying at room temperature for
a period of about 48 hours.
[0081] The resulting material had a porous, translucent, foam-like
appearance which differed from the collagenous material of Example
1.
[0082] Upon rehydration, the collagenous sheet material did not gel
or fragment, and had good structural integrity.
3. SEM Analysis
[0083] The collagenous material of Example 1 was examined by SEM.
Samples of approximately 7 m.times.7 mm were cut from the material
and mounted on SEM stubs using double-sided sticky tabs. Silver-dag
was used around the edges to reduce charge effects. The samples
were sputter coated with approximately 5 nm of gold/palladium. They
were viewed at 2 kV using a JEOL JSM-7500F scanning electron
microscope at a working distance of approximately 8 mm and the LEI
detector.
[0084] Results are shown in the scanning electron micrograph of
FIG. 1. It can be seen that, at least in part, the collagenous
material showed good retention of collagen structure and alignment
seen in the particulate collagen starting material.
4. Tensile Strength Testing
[0085] The tensile strength of the collagenous materials of
Examples 1 and 2 was tested using a Hounsfield tensiometer equipped
with a 1 kN load cell. Jaw speed was set at 10 mm/min, with samples
tested until failure. For reference, testing was repeated using
samples of Promogran.RTM., an existing wound care product
comprising a freeze-dried composite of oxidised regenerated
cellulose and collagen.
[0086] Testing was carried out using representative 100 mm.times.10
mm samples, fully hydrated in 0.9% saline. Maximum load before
failure was recorded to provide ultimate tensile strength in
Newtons.
[0087] The collagenous material of Example 1 was found to have a
tensile strength of around 0.3N in the hydrated state and the
collagenous material of Example 2 had a tensile strength of around
0.9N in the hydrated state. The hydrated Promogran.RTM. material
had a tensile strength of around 0.1 N.
[0088] It is of course to be understood that the invention is not
intended to be restricted by the details of the above specific
embodiments, which are provided by way of example only.
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